CN116726019A - Preparation method and application of indole diketopiperazine alkaloid - Google Patents
Preparation method and application of indole diketopiperazine alkaloid Download PDFInfo
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- CN116726019A CN116726019A CN202310557050.9A CN202310557050A CN116726019A CN 116726019 A CN116726019 A CN 116726019A CN 202310557050 A CN202310557050 A CN 202310557050A CN 116726019 A CN116726019 A CN 116726019A
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- ethyl acetate
- rice
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- fermentation
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- IRABMPNBAAQRLL-UHFFFAOYSA-N 1h-indole;piperazine-2,3-dione Chemical class O=C1NCCNC1=O.C1=CC=C2NC=CC2=C1 IRABMPNBAAQRLL-UHFFFAOYSA-N 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 15
- XNRPVPHNDQHWLJ-PMACEKPBSA-N tryprostatin A Chemical compound N1C(=O)[C@@H]2CCCN2C(=O)[C@@H]1CC1=C(CC=C(C)C)NC2=CC(OC)=CC=C21 XNRPVPHNDQHWLJ-PMACEKPBSA-N 0.000 claims abstract description 12
- 208000035150 Hypercholesterolemia Diseases 0.000 claims abstract description 8
- 239000000556 agonist Substances 0.000 claims abstract description 8
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- XNRPVPHNDQHWLJ-UHFFFAOYSA-N Tryprostatin A Natural products N1C(=O)C2CCCN2C(=O)C1CC1=C(CC=C(C)C)NC2=CC(OC)=CC=C21 XNRPVPHNDQHWLJ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 230000001105 regulatory effect Effects 0.000 claims abstract description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 57
- 238000000855 fermentation Methods 0.000 claims description 24
- 230000004151 fermentation Effects 0.000 claims description 24
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 22
- 241000209094 Oryza Species 0.000 claims description 19
- 235000007164 Oryza sativa Nutrition 0.000 claims description 19
- 235000009566 rice Nutrition 0.000 claims description 19
- 239000001963 growth medium Substances 0.000 claims description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 9
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- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 6
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- JXDYKVIHCLTXOP-UHFFFAOYSA-N isatin Chemical compound C1=CC=C2C(=O)C(=O)NC2=C1 JXDYKVIHCLTXOP-UHFFFAOYSA-N 0.000 abstract 1
- 150000004885 piperazines Chemical class 0.000 abstract 1
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- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 10
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- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 5
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 5
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 5
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
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- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
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- VTNULXUEOJMRKZ-UHFFFAOYSA-N 3-[4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy-N-(2H-tetrazol-5-ylmethyl)benzamide Chemical compound N=1NN=NC=1CNC(C1=CC(=CC=C1)OC1=NC(=CC(=C1)CN)C(F)(F)F)=O VTNULXUEOJMRKZ-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
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- 108090000943 Cholesterol 7-alpha-monooxygenases Proteins 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- ABRVLXLNVJHDRQ-UHFFFAOYSA-N [2-pyridin-3-yl-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound FC(C1=CC(=CC(=N1)C=1C=NC=CC=1)CN)(F)F ABRVLXLNVJHDRQ-UHFFFAOYSA-N 0.000 description 2
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- 125000004450 alkenylene group Chemical group 0.000 description 2
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- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 2
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- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- CPHRCQUGNAGVIB-FWJAZRMLSA-N 12,13-dihydroxyfumitremorgin C Chemical compound O=C1[C@@H]2CCCN2C(=O)[C@]2(O)[C@@H](O)C(C3=CC=C(C=C3N3)OC)=C3[C@H](C=C(C)C)N21 CPHRCQUGNAGVIB-FWJAZRMLSA-N 0.000 description 1
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- ZEEBGORNQSEQBE-UHFFFAOYSA-N [2-(3-phenylphenoxy)-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound C1(=CC(=CC=C1)OC1=NC(=CC(=C1)CN)C(F)(F)F)C1=CC=CC=C1 ZEEBGORNQSEQBE-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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Abstract
The invention discloses a preparation method and application of indole diketopiperazine alkaloid. In particular, the invention discloses indole dione shown in formula (I)Application of piperazine alkaloid 12R,13S-Dihydroxy fumitremorgin C and/or tryprostatin A in preparing LXR alpha agonist, and application of indole diketopiperazine alkaloid 12R,13S-Dihydroxyfumitremorgin C and/or tryprostatin A shown in (I) in preparing medicine for preventing and/or treating diseases regulated by LXR alpha agonist. The invention provides a new alternative compound for developing the medicine for preventing and treating the hypercholesterolemia, has important significance for developing the Chinese ocean-derived medicine,
Description
Technical Field
The invention belongs to the field of natural products, and particularly relates to a preparation method and application of indole diketopiperazine alkaloid.
Background
Hypercholesterolemia is a metabolic disease with abnormal lipid metabolism, which is manifested by abnormal elevation of cholesterol in blood, especially low-density lipoprotein cholesterol (low density lipoprotein cholesterol, LDL-C). Abnormal elevation of LDL-C is an independent risk factor for atherosclerosis, leading to the development of coronary heart disease and heart failure, and severely compromising human health. Atherosclerosis is the pathological basis of cardiovascular diseases, so the lipid-lowering treatment has great significance for preventing and treating cardiovascular diseases such as coronary heart disease and the like. Studies have shown that lowering total cholesterol (total cholesterol, TC) in plasma is effective in preventing coronary heart disease. In addition, lipid-lowering therapy is also one of important measures for clinically preventing and treating cerebral infarction.
LXR is a member of the nuclear receptor superfamily of ligand-activated transcription factors, and there are two subtypes lxrα and lxrβ. They act as cholesterol sensors in humans, their activation by mobilizing cholesterol from the periphery, promoting its hepatic excretion and limiting its absorption, reducing its cellular uptake, and enhancing its conversion to bile acids (in the mouse liver), thereby net eliminating cholesterol from the body and improving the plasma lipoprotein profile.
Since the first clone in the 90 s of the 20 th century, the Liver X Receptor (LXR) has been studied for about 30 years and has shown great potential for the treatment of metabolic diseases such as atherosclerosis, hypercholesterolemia and NAFLD. In addition, many clinical trials are underway for diseases such as diarrhea, cancer, atherosclerosis and atopic dermatitis. LXR activation maintains lipid balance, improves atherosclerosis, regulates immunity, and has anti-inflammatory activity.
Some natural compounds have been found to show promising therapeutic promise by targeting lxrα or by the lxrα pathway to alleviate hypercholesterolemia. The structure and biological activity of natural products are more complex and diverse than compounds, and thus many natural products have fewer side effects in exerting therapeutic effects.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a preparation method and application of indole diketopiperazine alkaloid. According to the invention, through screening LXR alpha ligand from marine microorganism natural products, two indole diketopiperazine alkaloids 12R,13S-Dihydroxyfumitremorgin C and tryprostatin A separated from rice fermentation culture of sponge co-epiphyte Aspergillus sp.SCSIO 41420 are obtained.
In a first aspect, the invention provides the use of an indole diketopiperazine alkaloid 12R,13S-Dihydroxyfumitremorgin C and/or tryprostatinA of formula (I) in the preparation of an LXR alpha agonist,
further, the indole diketopiperazine alkaloid shown in the formula (I) improves the expression level of ABCA1 and CYP7A1 genes.
In a second aspect, the invention provides the use of an indole diketopiperazine alkaloid 12R,13S-Dihydroxyfumitremorgin C and/or tryprostatinA of formula (I) for the preparation of a medicament for the prophylaxis and/or treatment of a disease modulated by an LXR alpha agonist.
Further, the diseases regulated by lxrα agonists include hypercholesterolemia and various diseases induced thereby, hypertriglyceridemia and various diseases induced thereby, mixed hyperlipidemia and various diseases induced thereby.
Further, the induced diseases include non-alcoholic fatty liver disease, atherosclerosis and cardiovascular disease.
Further, the cardiovascular diseases include prevention and treatment of coronary heart disease, cerebral apoplexy, myocardial infarction and ischemic kidney diseases.
The third aspect of the invention provides a preparation method of indole diketopiperazine alkaloid 12R,13S-Dihydroxyfumitremorgin C and tryprostatinA shown in formula (I), comprising the following steps:
(1) Preparing a rice fermentation culture of a sponge coanda fungus Aspergillus sp.SCSIO 41420, extracting the rice fermentation culture with ethyl acetate, and concentrating an ethyl acetate extract to obtain a supernatant fermentation liquid extract;
(2) The extract is subjected to normal phase silica gel chromatographic column, petroleum ether/ethyl acetate/methanol system gradient elution is adopted, petroleum ether/ethyl acetate gradient elution is carried out from the volume ratio of 100:0,90:10,80:20,60:40,20:80,10:90,0:100, and ethyl acetate/methanol gradient elution is carried out from the volume ratio of 99:1,98:2; and (3) carrying out gradient elution in a ratio of 95:5,90:10,85:15,80:20 and 50:50, collecting a component Fr4 eluted by petroleum ether/ethyl acetate in a ratio of 80:20 and 60:40, separating and purifying to obtain 12R,13S-Dihydroxyfumitremorgin C, collecting a component Fr6 eluted by petroleum ether/ethyl acetate in a ratio of 10:90 and 0:100, carrying out gradient elution on the component Fr6 by adopting methanol-water, collecting a component Fr6-9 eluted by methanol-water in a ratio of 60:40 and 80:20, and separating and purifying to obtain the tryprostatin A.
Further, the rice fermentation culture of the sponge coanda fungus Aspergillus sp.SCSIO 41420 is prepared by the following method:
A. streaking Aspergillus sp.SCSIO 41420 strain on MB solid culture medium plate for culturing to obtain spore, inoculating the spore into MA liquid seed culture medium, and shake culturing at 26deg.C for 4 days to obtain seed culture solution;
B. inoculating the cultured seed culture solution into the amplified fermentation rice culture medium under aseptic condition, and shake culturing at 26deg.C for 30 days to obtain fermentation culture of Aspergillus sp.SCSIO 41420 strain.
Further, the formula of the MB solid culture medium comprises the following components: 15g/L of malt extract powder, 24g/L of crude sea salt and 15g/L, pH 7.0.0 of agar;
the formula of the MA liquid seed culture medium is as follows: 15g/L of malt extract powder, 24g/L of crude sea salt and pH7.0;
the formula of the fermented rice culture medium is as follows: 800g/L of rice and 24g/L of crude sea salt.
Further, in step a spores are inoculated into 100 lma liquid seed medium;
in the step B, 10mL of the cultured seed culture solution is inoculated into 250mL of the amplified fermentation rice culture medium.
Compared with the prior art, the invention has the advantages that:
1. the invention uses a HepG2 cell in-vitro model, adopts qPCR real-time fluorescence quantitative technology and WB protein immunoblotting technology, and proves that the compounds 12R,13S-Dihydroxyfumitremorgin C and trypostatin A can effectively activate LXR alpha and induce the expression of the downstream target genes ABCA1 and CYP7A1 to be increased.
2. According to the invention, a high cholesterol cell model is constructed in vitro, and the lipid content in the high cholesterol cell model can be reduced by confirming that the compounds 12R,13S-Dihydroxyfumitremorgin C and tryprostatinA through oil red staining and intracellular TG and T-CHO content measurement.
3. The invention proves the LXRalpha agonism of the compounds 12R,13S-Dihydroxyfumitremorgin C and the tryprotatin A for the first time, and provides a new alternative compound for preparing the medicines for preventing and treating the hypercholesterolemia.
4. The raw material of the invention is derived from marine sponge epiphyte, is a brand new discovery of marine source compounds in the field of preventing and treating hypercholesterolemia, widens the field of view for treating diseases by marine source medicaments, and has wide development and application prospects.
Drawings
FIG. 1 is a diagram of Compound 1 1 H NMR(500MHz,DMSO-d 6 ) A map;
FIG. 2 is a diagram of Compound 1 13 C NMR(125MHz,DMSO-d 6 ) A map;
FIG. 3 is a diagram of Compound 2 1 H NMR(500MHz,DMSO-d 6 ) A map;
FIG. 4 is a diagram of Compound 2 13 C NMR(125MHz,DMSO-d 6 ) And (5) a map.
FIG. 5 shows qPCR results of compounds 1 and 2, which are the effects of compounds 1 and 2 on the mRNA levels of HepG2 cells LXRalpha, ABCA1 and CYP7A 1.
FIG. 6 shows the WB results of compounds 1 and 2, which are the effects of compounds 1 and 2 on levels of LXRalpha, ABCA1 and CYP7A1 proteins in HepG2 cells.
FIG. 7 shows the TG and T-CHO levels of compounds 1 and 2 in HepG2 high cholesterol model cells.
FIG. 8 shows the results of oil red staining of compounds 1 and 2 in HepG2 high cholesterol model cells.
The specific embodiment is as follows:
the following examples are further illustrative of the invention and are not intended to be limiting thereof. In the examples, each of the starting reagent materials is commercially available, and the experimental methods without specifying the specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.
Example 1:
this example provides the preparation and structural identification of compound 1 and compound 2 as shown in formula (I).
The preparation method of the compound 1 and the compound 2 comprises the following steps:
1. seed culture
(1) Seed medium formulation (MA medium): dissolving 15g of malt extract powder, 24g of crude sea salt and 1. 1L, pH 7.0.0 of distilled water according to the proportion, fully and uniformly mixing, packaging 100mL of the mixture into 250mL conical flasks, and sterilizing the mixture at 121 ℃ for 30 minutes to obtain a seed culture medium.
(2) Culturing seed liquid: the strain Aspergillus sp.SCSIO 41420 was streaked onto MB solid medium (malt extract powder 15g, crude sea salt 24g, agar 15g, distilled water 1L, pH 7.0.0) plates and cultured until sporulation was achieved, and then the spores were inoculated into MA liquid seed medium and cultured on a shaker at 26℃for 4 days at a rotation speed of 180rpm to obtain a seed culture solution.
2. Amplifying fermentation culture
(1) The formulation of the amplified fermentation medium comprises: 200g of rice, 6g of crude sea salt and 250mL of distilled water.
(2) Amplified fermentation culture under aseptic condition, 10mL of the cultured seed culture solution is respectively inoculated into amplified fermentation rice culture medium, and the amplified fermentation rice culture medium is subjected to shake culture at 26 ℃ for 30 days to obtain a fermentation culture of Aspergillus sp.SCSIO 41420.
3. Extraction and separation
Extraction of fermentation culture: after the fermentation is finished, adding an equal volume of ethyl acetate into each fermentation bottle, soaking for 1h, mashing rice, extracting with ethyl acetate for 3 times after ultrasonic treatment for 15min, collecting ethyl acetate supernatant, and volatilizing and concentrating into extract under a fume hood to obtain about 39g.
Separation of the compounds: dissolving the extract with a small amount of ethyl acetate, mixing the extract with 100-200 mesh silica gel, performing forward silica gel column chromatography, and performing gradient elution on petroleum ether/ethyl acetate/methanol system, wherein the petroleum ether-ethyl acetate is subjected to gradient elution from the volume ratio of 100:0,90:10,80:20,60:40,20:80,10:90 and 0:100, and the ethyl acetate-methanol is subjected to gradient elution from the volume ratio of 99:1 and 98:2; gradient elution of 95:5,90:10,85:15,80:20,50:50 yielded 13 fractions Fr.1-Fr.13. Collecting fraction Fr4 and Fr.4 of petroleum ether/ethyl acetate eluted from the volume ratio of 80:20 and 60:40, and purifying with semi-preparative high performance liquid to obtain compound 3 (4.48 mg, t) R 22min,70% acetonitrile/water, 2.5mL/min,210 nm). Collecting components Fr6 and Fr.6 eluted from petroleum ether/ethyl acetate in a volume ratio of 10:90 and 0:100, performing medium-pressure reverse silica gel column chromatography, and performing gradient elution by a methanol-water system to obtain 13 fractions Fr.6-1-Fr.6-13. Collecting fractions Fr6-9 and Fr.6-9 eluted with methanol-water volume ratio of 60:40 and 80:20, and purifying by semi-preparative HPLC to obtain compound 2 (4.90 mg, t) R 14min,35% acetonitrile/water, 2.5mL/min,210 nm).
4. Physicochemical data of Compounds 1 and 2
FIG. 1 is a diagram of Compound 1 1 H NMR(500MHz,DMSO-d 6 ) A map;
FIG. 2 is a diagram of Compound 1 13 C NMR(125MHz,DMSO-d 6 ) A map;
FIG. 3 is a diagram of Compound 2 1 H NMR(500MHz,DMSO-d 6 ) A map;
FIG. 4 is a diagram of Compound 2 13 C NMR(125MHz,DMSO-d 6 ) And (5) a map.
Physicochemical data for compound 1 and compound 2 are as follows:
compound 1 was a white powder. The compound is 1 H NMR spectrum showed 3 methyl [ delta ] H 1.92(s,3H,H-23),1.58(s,3H,H-24),3.75(s,3H,H-25)]3 methylene [ delta ] H 2.29(m,1H,H 2 -14),1.87(m,2H,H-15),1.87(m,1H,H 2 -14),3.51(m,1H,H 2 -16),3.46(m,1H,H 2 -16)]4 olefinic hydrogen protons [ delta ] H 4.76(m,1H,H-21),7.61(d,J＝8.7Hz,1H,H-5),6.62(dd,J＝8.7,2.3Hz,1H,H-6),6.82(d,J＝2.3Hz,1H,H-8)]3 methines [ delta ] H 5.75(dd,J＝9.5,1.1Hz,1H,H-10),4.39(dd,J＝9.5,6.9Hz,1H,H-13),5.51(dd,J＝3.5,1.1Hz,1H,H-20)]。 13 The C NMR spectrum showed 22 carbon signals including 3 methyl groups, 3 methylene groups, 4 alkenylene groups, 3 methine groups, 7 quaternary carbons, 2 carbonyl groups. Comparison by literature [1] Compound 3 was identified as 12R,13S-Dihydroxyfumitremorgin C.
Compound 2 was brown oily. The compound is 1 HNMR spectra showed 3 methyl [ delta ] H 1.69(s,3H,H-23),1.71(s,3H,H-24),3.72(s,3H,H-25)]5 methylene [ delta ] H 3.48(dd,J＝14.6,7.7Hz,1H,H 2 -10),2.96(dd,J＝14.6,6.1Hz,1H,H 2 -10),1.40(m,1H,H 2 -15),1.90(m,1H,H 2 -15),1.60(m,1H,H 2 -16),1.11(m,1H,H 2 -16),3.17(m,1H,H 2 -17),3.14(m,1H,H 2 -17),3.40(m,1H,H 2 -20),3.40(m,1H,H 2 -20)]4 olefinic hydrogen protons [ delta ] H 7.32(d,J＝8.0Hz,1H,H-5),6.57(dd,J＝8.0,2.3Hz,1H,H-6),6.77(d,J＝2.3Hz,1H,H-8),5.3(m,1H,H-21)]2 methines [ delta ] H 4.00(dd,J＝8.0,2.3Hz,1H,H-11),4.22(t,J＝5.7Hz,1H,H-14)]。 13 The C NMR spectrum showed 22 carbon signals including 3 methyl groups, 5 methylene groups, 4 alkenylene groups, 2 methine groups, 6 quaternary carbons, 2 carbonyl groups. Comparison by literature [2] DeterminingCompound 2 is tryprostatinA.
[1]Zhang DH,Noviendri D,Nursid M,Yang XD,Son BW*.12,13-dihyd roxyfumitremorgin C,fumitremorgin C,and brevianamide F,antibacterial diketo piperazine alkaloids from the marine-derived fungus Pseudallescheria sp.Natural Product Sciences.2007,13(3):251-254.
[2]Liu R,Li H,Yang J,Zhou F.Indole Diketopiperazines from Endophytic Fungus ofAstragalus membranaceus and Biological Evaluation.Chemistry ofNatural Compounds.2018,54(6):1196-1198.
Through nuclear magnetic resonance data analysis of the compounds, the compounds 1 and 2 are determined to be known compounds 12R,13S-Dihydroxyfumitremorgin C and tryprostatine A, and the structures are shown as the formula (I):
example 2:
this example examined the effect of compound 1-2 on lxrα and its downstream target genes ABCA1 and CYP7A1 in agonism in vitro in HepG2 cells and on lipid content in a high cholesterol cell model.
Experimental cells: the invention adopts HepG2 human hepatoblastoma cells, cultures the HepG2 human hepatoblastoma cells in a DMEM culture medium containing 10 percent of fetal calf serum and 1 percent of green streptomycin mixed solution, and places the HepG2 human hepatoblastoma cells in CO 2 Incubator (37 ℃,5% CO) 2 ) Culturing.
Abbreviations meaning: OA (Oleic acid); PA (Palmitic acid ); T-Cho (serum cholesterol); TG (triglyceride).
1. Effect of Compound 1 and Compound 2 on LXR alpha and its downstream target genes ABCA1 and CYP7A1 1.1 cellular intervention
Taking in vitro cultured HepG2 cells in logarithmic growth phase at 2×10 5 The density of each hole is inoculated into a six-hole plate, and after the cells are attached to the wall after being cultured for 24 hours, the cells are divided into three groups and added with different compounds for co-culture for 24 hours.
Control group: equivalent amount of DMSO was added for co-culture.
Compound 1 group: co-cultivation was performed by adding 25. Mu.M of Compound 1.
Group 2: co-cultivation was performed by adding 25. Mu.M of Compound 2.
1.2 detection of results indicators
To detect the agonism of lxrα and its downstream target genes ABCA1 and CYP7A1 at the transcriptional level, total RNA was extracted from HepG2 cells after 24 hours of co-culture, reverse transcribed into cDNA, and then the transcriptional level expression changes were detected by qPCR.
To detect the agonism of lxrα and its downstream target genes ABCA1 and CYP7A1 at the protein translation level, total protein was extracted from HepG2 cells after 24 hours of co-culture, and the protein translation level expression changes were detected by Western Blot.
1.3 experimental results
LXRalpha and the expression change of the downstream target genes ABCA1 and CYP7A1 are detected by qPCR. As shown in fig. 5, the mRNA expression amounts of lxrα were increased by 1.16-fold and 1.37-fold, respectively, whereas the mRNA levels of CYP7A1 and ABCA1 were significantly increased as expected by 5-fold, 5.02-fold and 2.05-fold, 2.13-fold, respectively. The data were statistically significant (P < 0.001).
LXRalpha and the expression change of the downstream target genes ABCA1 and CYP7A1 protein translation level are detected by utilizing WB. As shown in fig. 6, the protein expression amounts of lxrα were increased by 1.15-fold and 1.41-fold, respectively, whereas the mRNA levels of CYP7A1 and ABCA1 were significantly increased as expected by 2.32-fold, 1.69-fold and 1.91-fold, 3.04-fold, respectively. The data were statistically significant (P < 0.001).
The results showed that compounds 1 and 2, although not directly significantly increasing the expression level of lxrα at the transcriptional level and the translational level, significantly increased the expression level of the downstream target genes ABCA1 and CYP7 A1. Compounds 1 and 2 were demonstrated to increase their activity by binding to lxrα, agonizing their downstream target gene expression.
2. Effect of Compounds 1 and 2 on lipid content in high cholesterol cell models
2.1 high cholesterol cell model
Taking out the cultureHepG2 cells in the 2X 10 cell culture in the logarithmic growth phase 5 The density of each hole is inoculated into a cell climbing sheet or a six-hole plate, and after the cells are attached to the wall after being cultured for 24 hours, different compounds are added into four groups for co-culture for 48 hours.
Control group: the same amount of solvent (dimethyl sulfoxide) was added to conduct co-culture.
Model group: co-cultivation was performed by adding 0.5mM OA and 0.25mM PA.
Compound 1 group: co-cultivation was performed by adding 0.5mM OA and 0.25mM PA and 25. Mu.M Compound 1.
Group 2: co-cultivation was performed by adding 0.5mM OA and 0.25mM PA and 25. Mu.M Compound 2.
2.2 intracellular TG and T-CHO content determination
Adding standard substance, sample and control dd H according to Nanjing to build kit 2 After adding the corresponding reagent after O and after incubation at 37℃for 10min, the absorbance was measured at 510 nm.
2.3 cell oil Red staining
Re-heating and drying frozen slices, fixing in fixing solution for 15min, washing with tap water, and air drying.
Oil red staining: immersing the slices in oil red dye solution for 8-10min (shading with a cover), and washing with distilled water.
Background differentiation: slightly differentiating 75% alcohol, and washing with distilled water.
Hematoxylin staining: the slices are stained with hematoxylin dye solution for 3-5min, washed with running water, differentiated with differentiation solution, washed with running water, returned to blue, and washed with running water.
Sealing piece: the glycerogelatin tablet sealing tablet is sealed.
Microscopic examination, image acquisition and analysis.
2.4 experimental results
The results of intracellular TG and T-CHO content measurements are shown in FIG. 7. In the high cholesterol model, there was a significant decrease in intracellular triglyceride and total cholesterol levels following co-culture with compound 1 and 2 administration (data statistically significant).
The results of cell oil red staining are shown in figure 8. The number of red oil droplets in the high cholesterol cells co-cultured with compounds 1 and 2 was significantly reduced.
The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Those skilled in the art will appreciate that many variations, modifications, additions, or substitutions are possible, without departing from the scope of the invention as disclosed in the accompanying claims.
Claims (10)
1. The application of indole diketopiperazine alkaloid 12R,13S-Dihydroxyfumitremorgin C and/or tryprostatinA shown in the formula (I) in preparing LXR alpha agonist,
2. the use according to claim 1, wherein the indole diketopiperazine alkaloid of formula (I) increases the expression level of ABCA1 and CYP7A1 genes.
3. The application of indole diketopiperazine alkaloid 12R,13S-Dihydroxyfumitremorgin C and/or tryprostatinA shown in formula (I) in preparing medicaments for preventing and/or treating diseases regulated by LXRalpha agonists,
4. the use according to claim 3, wherein the disease modulated by lxrα agonists comprises hypercholesterolemia and induced diseases, hypertriglyceridemia and induced diseases, mixed hyperlipidemia and induced diseases.
5. The use according to claim 3, wherein said induced diseases include non-alcoholic fatty liver disease, atherosclerosis and cardiovascular diseases.
6. The use according to claim 5, wherein the cardiovascular disease comprises the prevention and treatment of coronary heart disease, cerebral stroke, myocardial infarction and ischemic kidney disease.
7. The preparation method of the indole diketopiperazine alkaloid 12R,13S-Dihydroxyfumitremorgin C and tryprostatine A shown in the formula (I) is characterized by comprising the following steps:
(1) Preparing a rice fermentation culture of a sponge coanda fungus Aspergillus sp.SCSIO 41420, extracting the rice fermentation culture with ethyl acetate, and concentrating an ethyl acetate extract to obtain a supernatant fermentation liquid extract;
(2) The extract is subjected to normal phase silica gel chromatographic column, petroleum ether/ethyl acetate/methanol system gradient elution is adopted, petroleum ether/ethyl acetate gradient elution is carried out from the volume ratio of 100:0,90:10,80:20,60:40,20:80,10:90,0:100, and ethyl acetate/methanol gradient elution is carried out from the volume ratio of 99:1,98:2; and (3) carrying out gradient elution in a ratio of 95:5,90:10,85:15,80:20 and 50:50, collecting a component Fr4 eluted by petroleum ether/ethyl acetate in a ratio of 80:20 and 60:40, separating and purifying to obtain 12R,13S-Dihydroxyfumitremorgin C, collecting a component Fr6 eluted by petroleum ether/ethyl acetate in a ratio of 10:90 and 0:100, carrying out gradient elution on the component Fr6 by adopting methanol-water, collecting a component Fr6-9 eluted by methanol-water in a ratio of 60:40 and 80:20, and separating and purifying to obtain the tryprostatin A.
8. The preparation method according to claim 7, wherein the rice fermentation culture of the sponge co-epiphyte Aspergillus sp.SCSIO 41420 is prepared by the following method:
A. streaking Aspergillus sp.SCSIO 41420 strain on MB solid culture medium plate, culturing to obtain spore, inoculating the spore into MA liquid seed culture medium, and shake culturing at 26deg.C for 4 days to obtain seed culture solution;
B. inoculating the cultured seed culture solution into the amplified fermentation rice culture medium under aseptic condition, and shake culturing at 26deg.C for 30 days to obtain fermentation culture of Aspergillus sp.SCSIO 41420 strain.
9. The method of claim 8, wherein the MB solid medium is formulated as follows: 15g/L of malt extract powder, 24g/L of crude sea salt and 15g/L, pH 7.0.0 of agar;
the formula of the MA liquid seed culture medium is as follows: 15g/L of malt extract powder, 24g/L of crude sea salt and pH7.0;
the formula of the fermented rice culture medium is as follows: 800g/L of rice and 24g/L of crude sea salt.
10. The method of claim 8, wherein in step a spores are inoculated into 100 lma liquid seed medium;
in the step B, 10mL of the cultured seed culture solution is inoculated into 250mL of the amplified fermentation rice culture medium.
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